This invention was made with government support under grants AI12103 and AM02255 awarded by the U.S. Department of Health and Human Services. The government has certain rights in the invention.
This invention relates to compositions and methods for preventing human diseases including autoimmune diseases. More particularly, this invention relates to nucleic acid sequences and amino acid sequences of Mycoplasma arthritidis mitogen (MAM), synthetic oligonucleotides and peptides having such sequences, a method of purifying MAM to electrophoretic and sequence homogeneity, and methods of using such sequences and purified MAM in preventing human disease.
In autoimmune disease, a breakdown of self-tolerance leads to generation of an immune response against a specific target antigen or antigens. Microbial agents have long been thought to trigger autoimmune diseases by possessing antigenic determinants that are crossreactive with antigens on target organs. More recently, it has been suggested that superantigens derived from bacteria, P. Marrack & J. Kappler, 248 Science 705 (1990); B. Fleischer, 10 Immunol. Today 262 (1989), mycoplasma, B. Cole & C. Atkin, 12 Immunol. Today 271 (1991), or viruses, W. Frankel et al., 349 Nature 526 (1991); P. Dyson et al., 349 Nature 531 (1991); Y. Choi et al., 350 Nature 203 (1991), may initiate autoimmune disease by activating specific anti-self T cell clones, J. White et al., 56 Cell 27 (1989); B. Cole et al., 144 J. Immunol. 425 (1990), X. Paliard et al., 253 Science 325 (1991), or by forming a superantigen bridge that crosslinks helper T (T.sub.H) cells with pre-immune B cells, thereby causing polyclonal B cell activation and secretion of autoimmune antibodies, S. Friedman et al., 34 Arthritis Rheum. 468 (1991), W. Mourad et al., 170 J. Exp. Med. 2011 (1989). In fact, recent studies have shown that MAM can trigger, enhance, and exacerbate experimental autoimmune collagen-induced arthritis (CIA). B. Cole & M. Griffiths, 36 Arthritis Rheum. 994 (1993).
Superantigens are potent mitogens that activate T cells by a unique pathway that binds the major histocompatibility complex (MHC) molecules on accessory cell or B lymphocyte surfaces with specific .beta.-chain variable regions (V.sub..beta.) of the .alpha./.beta. T cell receptor for antigen (TCR) present on T cells. Thus, a particular superantigen may be recognized by virtually all T cells that utilize a single or small group of TCR V.sub..beta. gene families. While there is some overlap, each superantigen is recognized by its use of a distinct and characteristic set of TCR V.sub..beta. gene families. Further, superantigens bind selectively and with high affinity to class II MHC molecules. In the absence of antigen processing and in a non-MHC-restricted manner, superantigen-class II MHC antigen complexes on the antigen-presenting cell surface trigger the proliferation of T cells expressing the relevant TCR V.sub..beta. gene products. Finally, the in vivo presence of superantigens profoundly alters the T cell repertoire. During the process of negative selection within the thymus, a superantigen clonally eliminates thymocytes with TCR that bear V.sub..beta. gene products that recognize exactly that superantigen. Superantigens include several staphylococcal enterotoxins, streptococcal pyrogenic exotoxins, a fragment of the group A streptococcus M protein, murine self antigens such as the Mls loci gene products (now known to be encoded by murine tumor retroviruses) and an unknown B cell-specific antigen, and Mycoplasma arthritidis T cell mitogen (MAM).
Mycoplasmas are the smallest self-replicating prokaryotes and are parasites of humans, birds, insects, plants, and virtually all other higher life forms. Mycoplasmas are the most common cause of naturally-occurring acute and chronic arthritis in many animal species. M. arthritidis is a naturally-occurring arthritogen of rodents that causes a chronic, relapsing disease that, histologically, closely resembles human rheumatoid arthritis. MAM was discovered when live organisms and culture supernatants of M. arthritidis were shown to induce the proliferation of, and elicit the differentiation of, cytolytic cells in mouse splenocytes. B. Cole et al., 127 J. Immunol. 1931 (1981). An insoluble, presumably membrane-bound B-cell mitogen was found to be associated with mycoplasma cells and was stable at 100.degree. C. In contrast, a soluble T-cell mitogen was present in culture supernatants and was heat labile at 56.degree. C. This heat labile T-cell mitogen is MAM. MAM was then shown to be a potent T-cell mitogen and inducer of gamma-interferon (IFN-.gamma.) for both murine and human lymphocytes. B. Cole et al., 128 J. Immunol. 2013 (1982); B. Cole & R. Thorpe, 131 J. Immunol. 2392 (1983); B. Cole & R. Thorpe, 43 Infect. Immun. 302 (1984); T. Moritz et al., 20 Scand. J. Immunol. 365 (1984); H. Kirchner et al., 20 Scand. J. Immunol. 133 (1984); H. Kirchner et al., 4 J. Interferon Res. 389 (1984).
MAM is produced to maximal titer in senescent broth cultures of M. arthritidis. Purification is difficult because MAM is produced in small amounts, is heat and acid (pH&lt;7.0) labile, and has great affinity for surfaces and large molecules, especially nucleic acids. Gel filtration of culture supernatants, at an ionic strength of about 0.5M, indicated that MAM has a molecular mass of about 15 kD whereas PAGE suggested the molecule was about 30 kD. C. Atkin et al., 137 J. Immunol. 1581 (1986); H. Kirchner et al., 24 Scand. J. Immunol. 245 (1986). Although Kirchner et al. claimed partial purification of MAM, their purification steps would have yielded .ltoreq.200-fold purification in the best of hands. Since their mitogenic assay was merely qualitative, they were unable to show yield or specific activity (mitogenicity per unit. protein). J. Homfeld et al., 7 Autoimmunity 317 (1990), have also described partial purification of MAM. Using a quantitative assay for MAM, C. Atkin et al., 137 J. Immunol. 1581 (1986), to achieve 200,000-fold purification. The calculated purification of MAM by the final gel filtration step implies measurement of protein, but the method was not given nor was a profile of protein or absorbance shown. Active fractions corresponded to the elution volumes of 15-20 kD standards, but no stainable protein by SDS-PAGE was identified nor was an amino acid sequence reported.
One of the major activities of MAM is its ability to cause a proliferation of lymphocytes from certain strains of mice, but not of others. Lymphocytes from BALB/c and C3H mice are readily activated whereas those of C57BL/10 mice fail to undergo proliferation in response to exposure to MAM. This negative or weak response of C57BL/10 mice enabled mapping one of the genes which control MAM reactivity to the I-E region of the murine H-2 MHC. Dependence upon MHC-bearing accessory cells for MAM-induced T-cell proliferation was consistent with this conclusion. This specificity for I-E bearing cells suggested that the I-E molecule might be the binding site for MAM. The fact that only splenocytes from I-E-bearing mouse strains could remove MAM activity from solution and liposomes with incorporated I-E, but not with I-A, molecules could present MAM to T cells supported this hypothesis. There is substantial evidence that the conserved a chain of the I-E molecule, or a combinatorial determinant between E.sub..alpha. and other .beta. chains, bears the MAM receptor. Evidence of this includes ATFR5 mice which lack E.sub..beta. respond to MAM through combinatorial E.sub..alpha. A.sub..beta. molecules, antibodies to a monoclonal antibody specific for E.sub..alpha. totally block MAM-induced proliferation, E.sub..alpha. transgenic mice on a C57BL/10 background present MAM, and transfected fibroblasts expressing E.sub..alpha. E.sub..beta. or E.sub..alpha. A.sub..beta. present MAM, whereas fibroblasts expressing A.sub..alpha. A.sub..beta. do not. B. Cole et al., 127 J. Immunol. 1931 (1981); B. Cole et al., 128 J. Immunol. 2013 (1982); B. Cole et al., 129 J. Immunol. 1352 (1982); B. Cole et al., 136 J. Immunol. 3572 (1986); M. Bekoff et al., 139 J. Immunol. 3189 (1987); M. Matthes et al., 18 Eur. J. Immunol. 1733 (1988); B. Cole et al., 144 J. Immunol. 420 (1990).
MAM, like other superantigens, is recognized by V.sub..beta. chain segments of the .alpha./.beta. TCR. This was demonstrated in progeny of test-crosses between RIIIS mice, which have massive deletions in their V.sub..beta. .alpha./.beta. T-cell repertoire, with (RIIIS.times.B10.RIII)F1 hybrids. B10.RIII mice contains all V.sub..beta. genes. Reactivity of lymphocytes with MAM cosegregated with expression of V.sub..beta. 8-bearing cells. Thus, at least the V.sub..beta. 8 TCR gene family was involved in recognition of MAM. In other experiments, clonal expansion of MAM-reactive BALB/c cells in vitro showed the activated cells expressed V.sub..beta. 8.1, V.sub..beta. 8.2, V.sub..beta. 8.3, and V.sub..beta. 6. MAM expansion of C57BR lymphocytes, which lack the V.sub..beta. 8 genes, resulted in strong expression of V.sub..beta. 6 in the activated population. Similarly, it has been shown that MAM can use TCRs expressing V.sub..beta. 5.1. These specificities of MAM for certain TCR genes was reported in B. Cole et al., 144 J. Immunol. 425 (1990); L. Baccala et al., 35 Arthritis Rheum. 434 (1992). In rats, MAM-reactive V.sub..beta. are homologous to the MAM-reactive V.sub..beta. of mice, with one exception. Engagement of rat V.sub..beta. 5.1, V.sub..beta. 6, V.sub..beta. 8.1, and V.sub..beta. 8.2, but not V.sub..beta. 8.3 were observed. In humans, the engaged V.sub..beta. included primarily V.sub..beta. 19.1 (alternatively termed V.sub..beta. 17.1) and, to a lesser extent, V.sub..beta. 3.1, V.sub..beta. 11.1, V.sub..beta. 12.1, and V.sub..beta. 13.1. R. Baccala et al., 35 Arthritis Rheum. 434 (1992). More recent experiments have shown that both genomic composition and allelic polymorphisms at the V.sub..beta. chain segment of the TCR exert profound effects upon the pattern of V.sub..beta. that are used by MAM. Thus, in V.sub..beta..sup.b haplotype mice, without genomic deletions of V.sub..beta. genes, V.sub..beta. 5.1, 6, 8.1, 8.2, and 8.3 engage MAM. In V.sub..beta..sup.a mice, with deletions in V.sub..beta. 5.1 to 5.3, 8.1 to 8.3, 9, 11, 12, and 13, there was significant expansion of V.sub..beta. 6-expressing cells and lesser expansions of V.sub..beta. 1-, 7-, and 16-expressing cells. In V.sub..beta..sup.c mice, with deletions of the same V.sub..beta. genes deleted in V.sub..beta..sup.a and additional deletions in V.sub..beta. 6, 15, and 17, there was a dominant expansion of V.sub..beta. 7 and V.sub..beta. 1, and a slight expansion of V.sub..beta. 3.1-expressing cells. B. Cole et al., 150 J. Immunol. 3291 (1993). Usage of V.sub..beta. 8 gene products is fairly common among other microbial superantigens, however V.sub..beta. 6 is only used by MAM and the Mls 1.sup.a antigen now known to be a murine retroviral superantigen.
MAM can also activate human peripheral blood lymphocytes; this reaction too is dependent upon MHC molecules. The human MHC HLA-DR molecule, the equivalent of the murine H2 I-E molecule, appears to possess the MAM binding site. Evidence of this includes inhibition of T-cell proliferation by anti-HLA-DR antibodies, production of IFN-.gamma. and induction of cytolytic cells in response to MAM, and presentation of MAM to human T-cells by cells transfected with I-E and the inhibition of the response by anti-I-E monoclonal antibodies. MAM can produce proliferation of human T-cells regardless of whether the cells express CD4 or CD8 molecules. TCR .alpha./.beta.-negative, .gamma./.delta.-positive cells also respond to MAM in the presence of appropriate accessory cells. R. Daynes et al., 129 J. Immunol. 936 (1982); B. Cole & R. Thorpe, 131 J. Immunol. 2392 (1983); M. Matthes et al., 18 Eur. J. Immunol. 1733 (1988); R. Baccala et al., 35 Arthritis Rheum. 434 (1992).
The response of human cells to MAM has always been found to be weaker than that of mouse cells and weaker than to lectin mitogens. In a direct comparison, human cells responded better to staphylococcal superantigens than to MAM, and mouse cells responded better to MAM. B. Fleischer et al., 146 J. Immunol. 11 (1991). This difference seems to issue from differences in the MHC/superantigen interaction since lymphocytes from transgenic mice expressing human MHC molecules respond better to staphylococcal superantigens than to MAM.
The apparent ability of individual superantigen molecules to interact simultaneously with MHC molecules on accessory cells and B cells, as well as with V.sub..beta. TCRs on T-cells, raises the possibility that superantigens might be able to initiate a B-T.sub.H cell collaboration resulting in polyclonal B cell activation. Peripheral blood lymphocytes from normal individuals or rheumatoid arthritis patients secreted significantly higher levels of IgG when co-cultured in vitro with MAM and pokeweed mitogen. Further, purified B cell cultures or B cells incubated with MAM-reactive T.sub.H cells failed to secrete significant levels of IgM. However, when B cells were briefly exposed to MAM or when MAM was added to B-T.sub.H cell mixtures, high levels of IgM were produced. This is important because abnormal B-T.sub.H cell interactions mimic the interaction seen in graft versus host disease that has been used as a model of systemic lupus erythematosus (SLE). In SLE, abnormal B cell reactivity results in production of a wide range of autoantibodies. P. Emery et al., 12 J. Rheumatol. 217 (1985); J. Tumang et al., 171 J. Exp. Med. 2153 (1990); S. Friedman et al., 34 Arthritis Rheum. 468 (1991).
M. arthritidis also causes a severe suppurative arthritis in rats which can also be associated with uveitis, C. Thirkill & D. Gregerson, 36 Infect. Immun. 775 (1982), conjunctivitis, urethritis, lethargy, and paralysis, J. Ward & R. Jones, 5 Arthritis Rheum. 163 (1962). MAM can activate rat lymphocytes. B. Cole et al., 36 Infect. Immun. 662 (1982). Splenic cells from inbred rat strains August, Buffalo, DA, Lewis, Wistar Furth, and (LEW x BN)F1 all responded well to MAM, but BN and MAXX rats responded very weakly or not at all. Genetic analysis showed that non-RT1 genes control responsiveness to MAM. Both responder and nonresponder splenic cells could bind MAM. These results contrast with the results obtained with non-responder mouse strains, wherein the cells failed to bind MAM due to the absence of the E.sub..alpha. chain of the I-E molecule. B. Cole et al., 129 J. Immunol. 1352 (1982); B. Cole et al., 136 J. Immunol. 2364 (1986).
Interestingly, the genetics of MAM-induced activation of rat lymphocytes resembles that of susceptibility to two experimentally-induced autoimmune diseases, experimental allergic encephalomyelitis (EAE) and collagen-induced arthritis (CIA). Thus, (LEW x BN)F1 rats are susceptible to both EAE and CIA, and responsiveness to MAM is a dominant trait, whereas (DA x BN)F1 rats are resistant to both EAE and CIA, and responsiveness to MAM is recessive. In both EAE and CIA, T-cells expressing V.sub..beta. TCRs are involved in disease pathogenesis. Since rat and mouse V.sub..beta. TCRs are quite similar, it is not surprising that MAM also activates rat V.sub..beta. 8-bearing T-cells. L. Baccala et al., 35 Arthritis Rheum. 434 (1992).
Importantly, this similarity between the genetic predisposition to CIA and lymphocyte reactivity to MAM is now known to be due to involvement of similar V.sub..beta. chain segments of the TCR on T cell surfaces. Thus, T cells bearing V.sub..beta. 6, V.sub..beta. 7. and V.sub..beta. 8 participate in CIA. T. Haqqi et al., 89 Proc. Nat'l Acad. Sci USA 1253 (1992). These same V.sub..beta. TCRs are also activated by MAM, B. Cole et al., 150 J. Immunol. 3291 (1993), thus presenting a mechanism whereby superantigens might activate autoimmune disease. In fact, recent studies, B. Cole & M. Griffiths, 36 Arthritis Rheum. 944 (1993), have demonstrated that the intravenous injection of MAM (1) into mice suboptimally immunized with collagen causes a triggering of arthritis, (2) into mice convalescing from CIA results in a flare of disease activity, and (3) into mice just after injection of collagen causes an acceleration of the development of arthritis.
MAM is also thought to play a role in the pathogenicity of M. arthritidis by causing immunosuppression of the host. M. arthritidis is frequently harbored in the respiratory tract of apparently healthy mice and rats. Its presence may be undetectable without extensive culturing since an antibody response may not be present. M. Davidson et al., 8 Curr. Microb. 205 (1983). Even in experimentally-injected mice and rats, where complement-fixing antibodies are rapidly produced, the immune response to M. arthritidis is defective. Neutralizing or growth-inhibiting antibodies, which play a major role in the control of mycoplasma infections, are not produced against M. arthritidis in rodents. Opsonizing antibodies are likewise not produced. Probably for these reasons, mycoplasmemia persists for up to 3 weeks in the peripheral circulation of intravenously-injected animals. B. Cole et al., 98 J. Bacteriol. 930 (1969); B. Cole & J. Ward, 7 Infect. Immun. 691 (1973); B. Cole & J. Ward, 8 Infect. Immun. 199 (1973).
MAM may be responsible for depressed host defenses. Mycoplasmas are cleared faster from the peripheral circulation of mouse strains which lack functional I-E molecules than from strains possessing I-E. B. Cole et al., 41 Infect. Immun. 1010 (1983). Lymphocytes taken from I-E-bearing mice injected intravenously with MAM exhibit an impaired ability to proliferate in response to MAM, and, to a lesser extent, to lectin mitogens. B. Cole & D. Wells, 58 Infect. Immun. 228 (1990). MAM also appears to suppress other T-cell functions, such as contact sensitivity to dinitrofluorobenzene (DNFB) and can prolong skin grafts across H-2 and non-H-2 barriers. In contrast, MAM appears not to have any consistent suppressive effect in vivo on B-cell functions, but, instead, enhances B-cell activity.
MAM also appears at least partially responsible for reactions involving toxicity and necrosis in experimentally-injected mice. One of the earliest symptoms following intravenous injection of large numbers of M. arthritidis is a toxic shock syndrome that is analogous to the human condition caused by a staphylococcal superantigen. Symptoms include lethargy, ruffled fur, conjunctivitis, fecal impaction, and death in some individuals. These effects were H-2 restricted in that animals with MAM-reactive lymphocytes were susceptible, whereas animals with MAM-nonreactive lymphocytes were resistant. In part, this reaction may be due to liberation of lymphokines and other inflammatory molecules mediated by MAM-induced activation of lymphocytes and macrophages since large doses of highly purified MAM yielded a similar toxic syndrome, but of much lesser duration and severity. B. Cole et al., 41 Infect. Immun. 1010 (1983); B. Cole & D. Wells, 58 Infect. Immun. 228 (1990).
MAM also appears to play a role in dermal necrosis induced by subcutaneous injection of M. arthritidis in susceptible animals. Susceptible mice possess functional I-E, whereas mice lacking functional I-E developed a suppurative abscess but without dermal damage. B. Cole et al., 85 J. Invest. Dermatol. 357 (1985).